Dileucine requires essential amino acids to stimulate mTORC1 signaling and muscle protein synthesis in C2C12 myotubes

Dileucine ingestion, a leucine dipeptide, has been shown to stimulate greater muscle protein synthesis rates compared to the ingestion of free leucine in vivo in humans. Therefore, the purpose of this study was to explore the potential mechanism for the anabolic effects of dileucine by examining canonical mTORC1 signaling cascade in vivo and in vitro . Consistent with previous observations with leucine alone and complete essential amino acids (EAA), we observed increased peripheral compared to central signaling intensity of p-RPS6 S240/244 after dileucine ingestion in human skeletal muscle (main effect P=0.048), but non-significant changes to total staining (P=0.0856), peripheral staining intensity (P=0.0729), or central staining intensity (P=0.0927). To further probe this potential anabolic mechanism, experiments in C2C12 myotubes revealed that leucine, but not dileucine, alone stimulated mTORC1 signaling and protein synthesis (puromycin incorporation). However, in the presence of other EAA, dileucine stimulated mTORC1 signaling (S6K1 T389 phosphorylation) and protein synthesis, as did leucine. Pharmacological inhibition of leucine transporter LAT1 did not influence leucine and dileucine-stimulated mTORC1 signaling and protein synthesis in the presence of EAA. We demonstrate that stimulation of p-RPS6 peripheral:central ratio in vivo by dileucine ingestion may be facilitated by the presence of EAA as in vitro experiments showed a small additive effect on mTORC1 signaling compared to EAA in the absence of leucine. When combined with EAA, dileucine was not statistically different from leucine at eliciting mTORC1 signaling or protein synthesis in vitro .